Stem cell competition driven by the Axin2-p53 axis controls brain size during murine development

Developmental cell

2023 Apr 06

Sun, XL;Chen, ZH;Guo, X;Wang, J;Ge, M;Wong, SZH;Wang, T;Li, S;Yao, M;Johnston, LA;Wu, QF;
PMID: 37054704 | DOI: 10.1016/j.devcel.2023.03.016

Cell competition acts as a quality-control mechanism that eliminates cells less fit than their neighbors to optimize organ development. Whether and how competitive interactions occur between neural progenitor cells (NPCs) in the developing brain remains unknown. Here, we show that endogenous cell competition occurs and intrinsically correlates with the Axin2 expression level during normal brain development. Induction of genetic mosaicism predisposes Axin2-deficient NPCs to behave as "losers" in mice and undergo apoptotic elimination, but homogeneous ablation of Axin2 does not promote cell death. Mechanistically, Axin2 suppresses the p53 signaling pathway at the post-transcriptional level to maintain cell fitness, and Axin2-deficient cell elimination requires p53-dependent signaling. Furthermore, mosaic Trp53 deletion confers a "winner" status to p53-deficient cells that outcompete their neighbors. Conditional loss of both Axin2 and Trp53 increases cortical area and thickness, suggesting that the Axin2-p53 axis may coordinate to survey cell fitness, regulate natural cell competition, and optimize brain size during neurodevelopment.

Identification of visual cortex cell types and species differences using single-cell RNA sequencing

Nature communications

2022 Nov 12

Wei, JR;Hao, ZZ;Xu, C;Huang, M;Tang, L;Xu, N;Liu, R;Shen, Y;Teichmann, SA;Miao, Z;Liu, S;
PMID: 36371428 | DOI: 10.1038/s41467-022-34590-1

The primate neocortex exerts high cognitive ability and strong information processing capacity. Here, we establish a single-cell RNA sequencing dataset of 133,454 macaque visual cortical cells. It covers major cortical cell classes including 25 excitatory neuron types, 37 inhibitory neuron types and all glial cell types. We identified layer-specific markers including HPCAL1 and NXPH4, and also identified two cell types, an NPY-expressing excitatory neuron type that expresses the dopamine receptor D3 gene; and a primate specific activity-dependent OSTN + sensory neuron type. Comparisons of our dataset with humans and mice show that the gene expression profiles differ between species in relation to genes that are implicated in the synaptic plasticity and neuromodulation of excitatory neurons. The comparisons also revealed that glutamatergic neurons may be more diverse across species than GABAergic neurons and non-neuronal cells. These findings pave the way for understanding how the primary cortex fulfills the high-cognitive functions.

Developmental single-cell transcriptomics of hypothalamic POMC neurons reveal the genetic trajectories of multiple neuropeptidergic phenotypes

eLife

2022 Jan 19

Yu, H;Rubinstein, M;Low, MJ;
PMID: 35044906 | DOI: 10.7554/eLife.72883

Proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus are essential to regulate food intake and energy balance. However, the ontogenetic transcriptional programs that specify the identity and functioning of these neurons are poorly understood. Here, we use single-cell RNA-sequencing (scRNA-seq) to define the transcriptomes characterizing Pomc-expressing cells in the developing hypothalamus and translating ribosome affinity purification with RNA-sequencing (TRAP-seq) to analyze the subsequent translatomes of mature POMC neurons. Our data showed that Pomc-expressing neurons give rise to multiple developmental pathways expressing different levels of Pomc and unique combinations of transcription factors. The predominant cluster, featured by high levels of Pomc and Prdm12 transcripts, represents the canonical arcuate POMC neurons. Additional cell clusters expressing medium or low levels of Pomc mature into different neuronal phenotypes featured by distinct sets of transcription factors, neuropeptides, processing enzymes, cell surface, and nuclear receptors. We conclude that the genetic programs specifying the identity and differentiation of arcuate POMC neurons are diverse and generate a heterogeneous repertoire of neuronal phenotypes early in development that continue to mature postnatally.

A harmonized atlas of mouse spinal cord cell types and their spatial organization

Nature communications

2021 Sep 29

Russ, DE;Cross, RBP;Li, L;Koch, SC;Matson, KJE;Yadav, A;Alkaslasi, MR;Lee, DI;Le Pichon, CE;Menon, V;Levine, AJ;
PMID: 34588430 | DOI: 10.1038/s41467-021-25125-1

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

An MST4-pβ-CateninThr40 Signaling Axis Controls Intestinal Stem Cell and Tumorigenesis

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

2021 Jul 08

Zhang, H;Lin, M;Dong, C;Tang, Y;An, L;Ju, J;Wen, F;Chen, F;Wang, M;Wang, W;Chen, M;Zhao, Y;Li, J;Hou, SX;Lin, X;Hu, L;Bu, W;Wu, D;Li, L;Jiao, S;Zhou, Z;
PMID: 34240584 | DOI: 10.1002/advs.202004850

Elevated Wnt/β-catenin signaling has been commonly associated with tumorigenesis especially colorectal cancer (CRC). Here, an MST4-pβ-cateninThr40 signaling axis essential for intestinal stem cell (ISC) homeostasis and CRC development is uncovered. In response to Wnt3a stimulation, the kinase MST4 directly phosphorylates β-catenin at Thr40 to block its Ser33 phosphorylation by GSK3β. Thus, MST4 mediates an active process that prevents β-catenin from binding to and being degraded by β-TrCP, leading to accumulation and full activation of β-catenin. Depletion of MST4 causes loss of ISCs and inhibits CRC growth. Mice bearing either MST4T178E mutation with constitutive kinase activity or β-cateninT40D mutation mimicking MST4-mediated phosphorylation show overly increased ISCs/CSCs and exacerbates CRC. Furthermore, the MST4-pβ-cateninThr40 axis is upregulated and correlated with poor prognosis of human CRC. Collectively, this work establishes a previously undefined machinery for β-catenin activation, and further reveals its function in stem cell and tumor biology, opening new opportunities for targeted therapy of CRC.

Hedgehog signaling promotes expansion of Meibomian Gland stem cells in vivo

Investigative Ophthalmology & Visual Science

2022 Jan 01

Zhu, X;Xu, M;Grachtchouk, M;

RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells. Transcriptional profiling of GLI2ΔN-expressing and control MGs revealed that forced GLI2ΔN expression caused greatly increased expression of _Lrig1_ and _Lgr6_ and suppressed expression of meibocyte differentiation genes.

Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.

Nat Cell Biol.

2017 Apr 10

Turco MY, Gardner L, Hughes J, Cindrova-Davies T, Gomez MJ, Farrell L, Hollinshead M, Marsh SG, Brosens JJ, Critchley HO, Simons BD, Hemberger M, Koo BK, Moffett A, Burton GJ.
PMID: 28394884 | DOI: 10.1038/ncb3516

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.

Engineered Wnt ligands enable blood-brain barrier repair in neurological disorders

Science (New York, N.Y.)

2022 Feb 18

Martin, M;Vermeiren, S;Bostaille, N;Eubelen, M;Spitzer, D;Vermeersch, M;Profaci, CP;Pozuelo, E;Toussay, X;Raman-Nair, J;Tebabi, P;America, M;De Groote, A;Sanderson, LE;Cabochette, P;Germano, RFV;Torres, D;Boutry, S;de Kerchove d'Exaerde, A;Bellefroid, EJ;Phoenix, TN;Devraj, K;Lacoste, B;Daneman, R;Liebner, S;Vanhollebeke, B;
PMID: 35175798 | DOI: 10.1126/science.abm4459

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful blood-borne factors. Although BBB dysfunction is a hallmark of several neurological disorders, therapies to restore BBB function are lacking. An attractive strategy is to repurpose developmental BBB regulators, such as Wnt7a, into BBB-protective agents. However, safe therapeutic use of Wnt ligands is complicated by their pleiotropic Frizzled signaling activities. Taking advantage of the Wnt7a/b-specific Gpr124/Reck co-receptor complex, we genetically engineered Wnt7a ligands into BBB-specific Wnt activators. In a "hit-and-run" adeno-associated virus-assisted CNS gene delivery setting, these new Gpr124/Reck-specific agonists protected BBB function, thereby mitigating glioblastoma expansion and ischemic stroke infarction. This work reveals that the signaling specificity of Wnt ligands is adjustable and defines a modality to treat CNS disorders by normalizing the BBB.

Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells.

Science.

2018 Feb 01

Nabhan A, Brownfield DG, Harbury PB, Krasnow MA, Desai TJ.
PMID: 29420258 | DOI: 10.1126/science.aam6603

Alveoli, the lung's respiratory units, are tiny sacs where oxygen enters the bloodstream. They are lined by flat AT1 cells, which mediate gas exchange, and AT2 cells, which secret surfactant. Rare AT2s also function as alveolar stem cells. We show that AT2 lung stem cells display active Wnt signaling and many of them are near single, Wnt-expressing fibroblasts. Blocking Wnt secretion depletes these stem cells. Daughter cells leaving the Wnt niche transdifferentiate into AT1s: maintaining Wnt signaling prevents transdifferentiation whereas abrogating Wnt signaling promotes it. Injury induces AT2 autocrine Wnts, recruiting 'bulk' AT2s as progenitors. Thus, individual AT2 stem cells reside in single cell fibroblast niches providing juxtacrine Wnts that maintain them, whereas injury induces autocrine Wnts that transiently expand the progenitor pool. This simple niche maintains the gas exchange surface, and is coopted in cancer.

Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells

Stem cell reports

2023 Mar 07

Miwata, T;Suga, H;Kawaguchi, Y;Sakakibara, M;Kano, M;Taga, S;Soen, M;Ozaki, H;Asano, T;Sasaki, H;Miyata, T;Yasuda, Y;Kobayashi, T;Sugiyama, M;Onoue, T;Takagi, H;Hagiwara, D;Iwama, S;Arima, H;
PMID: 36963388 | DOI: 10.1016/j.stemcr.2023.02.006

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

Gut-brain communication by distinct sensory neurons differently controls feeding and glucose metabolism

Cell metabolism

2021 May 21

Borgmann, D;Ciglieri, E;Biglari, N;Brandt, C;Cremer, AL;Backes, H;Tittgemeyer, M;Wunderlich, FT;Brüning, JC;Fenselau, H;
PMID: 34043943 | DOI: 10.1016/j.cmet.2021.05.002

Sensory neurons relay gut-derived signals to the brain, yet the molecular and functional organization of distinct populations remains unclear. Here, we employed intersectional genetic manipulations to probe the feeding and glucoregulatory function of distinct sensory neurons. We reconstruct the gut innervation patterns of numerous molecularly defined vagal and spinal afferents and identify their downstream brain targets. Bidirectional chemogenetic manipulations, coupled with behavioral and circuit mapping analysis, demonstrated that gut-innervating, glucagon-like peptide 1 receptor (GLP1R)-expressing vagal afferents relay anorexigenic signals to parabrachial nucleus neurons that control meal termination. Moreover, GLP1R vagal afferent activation improves glucose tolerance, and their inhibition elevates blood glucose levels independent of food intake. In contrast, gut-innervating, GPR65-expressing vagal afferent stimulation increases hepatic glucose production and activates parabrachial neurons that control normoglycemia, but they are dispensable for feeding regulation. Thus, distinct gut-innervating sensory neurons differentially control feeding and glucoregulatory neurocircuits and may provide specific targets for metabolic control.

Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis.

Nature

2017 Aug 16

Sigal M, Logan CY, Kapalczynska M, Mollenkopf HJ, Berger H, Wiedenmann B, Nusse R, Amieva MR, Meyer TF.
PMID: 28813421 | DOI: 10.1038/nature23642

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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